We have made progress in our goal of defining germline modifiers of tumor progression and metastasis in prostate cancer. First, we are utilizing a number of well characterized mouse models of prostate cancer to investigate the role of hereditary factors in metastasis. Specifically, we are in the process of mapping prostate tumor progression and metastasis quantitative trait loci (QTLs) by using F2 intercross strategies and various recombinant inbred (RI) mouse strains. We are primarily utilizing the TRAMP model of prostate tumorigenesis, a model that recapitulates many of the features seen in aggressive neuroendocrine prostate cancer. We have initially approached this by crossing TRAMP mice to the eight progenitor strains of the Collaborative Cross (CC) RI panel. The goal here is to prove that the introduction of germline polymorphism through breeding influences prostate tumor progression and metastasis in mice. We have observed profound differences in tumor burden and metastasis frequencies in each of the TRAMP x CC progenitor F1 strains. We are following up on this initial observation by performing F2 intercrosses with the strains that displayed the greatest phenotypic differences compared to the wildtype TRAMP mouse. However, the molecular and histological features observed in tumors derived from commonly used mouse models of prostate cancer are somewhat different from those observed in human prostate cancer. We are therefore in the process of developing a novel prostate cancer mouse model. The aim here will be to develop an animal model that more faithfully recapitulates the molecular events that lead to the initiation of tumorigenesis in human prostate cancer. Specifically, a transgenic mouse is being generated that over-expresses micro-RNAs (miRNAs) targeting genes that are dysregulated in the early stages of prostate tumorigenesis. We have completed synthesis of these transgenic miRNA constructs and are in the early stages of introducing them into the germline of laboratory mice. We are also continuing to characterize the role of Rrp1b (Ribosomal RNA Processing 1 Homolog B) in tumor progression and metastasis in breast cancer. Our previous work has demonstrated that dysregulation of RRP1B induces profound changes in gene expression in in vitro and in vivo models. These profound effects upon gene expression are likely a consequence of a direct or indirect interaction between RRP1B and chromatin. Additionally, a polymorphism was identified within human RRP1B (G1421A;P436L), which is associated with differential survival in multiple breast cancer cohorts. We are therefore characterizing the function of this gene by using transgenic Rrp1b models as well as by developing an Rrp1b knockout mouse. Through the use of a transgenic Rrp1b mouse model we have confirmed that Rrp1b is indeed a metastasis suppressor. We expect to strengthen these observations using an Rrp1b conditional knockout, the production of which is nearing completion. Additionally, Rrp1b expression is being dysregulated in a number of manners in breast cancer cell lines, and we are assessing the impact of this on the in vitro and in vivo behaviors of these cells. This year, we have made substantial progress on this aspect of our work on RRP1B, which has given us greater insight into the function of this protein. We have previously shown that RRP1B interacts with a variety of nucleosome binding proteins (e.g. PARP1, Histone H1X), which may account for the effects that it has upon global gene expression. Additionally, we have previously demonstrated that RRP1B physically interacts with a number of proteins involved in the regulation of alternative mRNA splicing (e.g. SF2/ASF, SRP55). This year we have performed ChIP-seq and mRNA-Seq to investigate the role of endogenous RRP1B in both of these processes. We have subsequently been able to define genomic regions bound by endogenous RRP1B, which is helping us clarify its role in the regulation of gene expression. Also, our preliminary analyses demonstrate that RRP1B does indeed modulate alternative mRNA splicing, a process which is ubiquitously dysregulated in advanced tumorigenesis.